Clostridium difficile infection (CDI) is now the most common definable cause of hospital-acquired and antibiotic-associated diarrhea in the United States, with the total cost of treatment estimated between 1 to 4.8 billion U.S. dollars annually. C. difficile (CD), a multidrug-resistant Gram-positive anaerobic pathogen, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. Thus, antibiotic therapy is a major risk factor for CDI. Treatment has been hampered by increased virulence of the causative strains, sporulation, recurrence, and antibiotics used in treatment that disrupt the composition and colonization resistance of the colonic microbiota. As a result, there is an urgent need for non-antibiotic treatments that preserve the colonic microbiota. This study will evaluate repurposed FDA-approved salicylate-based drugs as novel non-antibiotic treatments for CDI without altering the gut microbiota. Pathogenic CD strains produce toxins A and B, which are directly responsible for disease because only strains that produce either of these toxins cause disease. Therefore, inhibiting toxin synthesis or toxin activity, which directly targets the cause f colonic injury and illness, is a promising approach to combat CDI. We have established that the CD toxins are stringently regulated by quorum signaling and have discovered that certain FDA-approved salicylate-based drugs currently used to treat pain and inflammation inhibit toxin synthesis in both hypervirulent and non-hypervirulent CD strains. We have also identified indole as a potent inhibitor of CD toxin activity. Indole is primarily produced by the gut microbiota as a end product of tryptophan metabolism. None of these compounds affect CD growth at their toxin-inhibitory concentrations. These are the first inhibitors of CD toxin synthesis identified an demonstrate potential as promising non-antibiotic therapeutic agents against CDI. Our central hypothesis is that these inhibitors block the toxin synthesis regulatory pathway, thereby preventing the transcription of the toxin genes. Here, we will (i) identify the target and the mechanism of inhibition of these salicylate-based inhibitors, (ii) evaluate their in vitro efficacy against clinical CD isolates, and (iii) examine the efficacy of a cocktail of these toxin synthesis and toxin activity inhibitors as a combination therapy in preventing CDI-induced inflammation, colonic pathology, and recurrence in a CDI model. At the conclusion of this project, new knowledge about CD toxin regulation and pathogenesis will be established and novel treatment methods identified. Specifically, we will gain a new understanding of the target and mechanism of inhibition of these CD toxin inhibitors and most importantly, a new and innovative therapeutic strategy using a cocktail of both non-antibiotic toxin synthesis and toxin activity inhibitors as a combination therapy to combat this major public health problem. These preclinical studies will form the basis for future clinical trials to evaluate the use of these potent toxin inhibitors for he treatment of CDI. Our department, Center for Infectious Diseases at the University of Texas School of Public Health is engaged in multidisciplinary studies with on-going clinical trials of CD treatments and prevention. Thus, we are strategically situated with access to CDI patient population to evaluate these inhibitors and to conduct phases I and II clinical trials to test for he efficacy of this novel non- antibiotic therapy.